Separation, purification, partial characterization and comparison of the heavy and light chains of botulinum neurotoxin types A, B, and E

Clostridium botulinum produces botulinum neurotoxin (NT) in antigenically distinct forms. When isolated from bacterial cultures type E is a single chain, type B is a mixture of single and two-chain molecules, and type A is essentially a two-chain molecule (Mr approximately 150,000). Protease(s) in the cultures or trypsin nick single-chain NT to the two-chain form. The heavy (Mr approximately 100,000) and light (Mr approximately 50,000) chains of the two-chain molecule remain held together by -S-S-bond(s). The two chains are presumed to have different functions. NT binds to nerve cells via the heavy chain and then light chain enters the cell and blocks release of acetylcholine (Simpson, L. L. (1981) Pharmacol. Rev. 33, 155-188). We nicked single-chain NT to form the two-chain form with trypsin, minimizing secondary cleavages, then separated and purified the heavy and light chains using ion-exchange chromatography. The technique, with minor modifications, is a generalized method for types A, B, and E. These subunit chains (each a single band in sodium dodecyl sulfatepolyacrylamide gel electrophoresis) were analyzed for their complete amino acid compositions. The amino acid contents of the heavy and light chains agreed well with the parent two-chain molecule. This affirms that NT is composed of two chains. The two subunit chains are now usable for amino acid sequence and other studies. Comparison of the amino acid contents indicates more similarity among the light chains than the heavy chains of the three NT types, a similarity that agrees with our published partial amino acid sequences (first 13-18 residues) of these chains. Several (up to 9) different amino acid residues of the heavy chain (which is twice the size of the light chain) are present in double the number of corresponding residues in the light chain.     

Partial amino acid sequences of the heavy and light chains of botulinum neurotoxin type E

The dichain type E botulinum neurotoxin, a product of nicking the single chain protein by trypsin, is composed of a heavy and light chains. Sequence of the first 13 and 20 N-terminal residues of these two chains were determined. Also, proof is provided here that (i) the light chain of the nicked (dichain) is derived from the N-terminal one-third of the parent single chain neurotoxin, and (ii) molecular events leading to the activation, of the single chain neurotoxin cannot involve tryptic cleavage at or very close to the N-terminal of the single chain protein. The partial amino acid sequence of the light chain of botulinum type E and tetanus neurotoxins show significant similarity between the two clostridial neurotoxins.     

Botulinum neurotoxin type B (strain 657): partial sequence and similarity with tetanus toxin

The type B neurotoxin (NT) isolated from Clostridium botulinum (strain 657) behaved as a mixture of single (unnicked) and dichain (nicked) proteins, both of Mr approximately 150 kDa. When the dichain NT was reduced by mercaptoethanol, the two chains migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as separate polypeptides of Mr approximately 100 and 50 kDa that appeared similar to the heavy and light chains of other serotypes of botulinum NT. The N-terminal amino acid sequences of the two chains were determined. They were as follows: light chain: Pro-Val-Thr-Ile-Asn-Asn-Phe-Asn-Tyr-Asn-Asp-Pro-Ile-Asp-Asn-Asn-Asn-Ile- Ile-Met - Met-Glu-Pro-Pro-Phe-Ala-Arg-Gly-Met-Gly-Arg-Tyr-Tyr-Lys-Ala-Phe-Lys-Ile- Thr-Asp - Arg-Ile-Trp-Ile-; and heavy chain: Ala-Pro-Gly-Ile-X-Ile-Asp-Val-Asp-Asn-Glu-Asp-Leu-Phe-Phe-Ile-Ala-Asp-Ly s-Asn- Ser-Phe-Arg-Asp-Asp-Leu-. These two sequences matched exactly with those of the light and heavy chains of type B NT (strain Okra) of which only 16 and 18 residues were known (J. Biol. Chem. (1985) 260, 10461). The above sequences were different from those of type A NT. Immunoprecipitation reactions of type B NT isolated from strains 657 and Okra were indistinguishable against polyclonal anti-type B NT serum. These two preparations did not produce precipitin reactions with polyclonal anti-type A NT serum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolated light chains of botulinum neurotoxins inhibit exocytosis. Studies in digitonin-permeabilized chromaffin cells

The effects of botulinum neurotoxins or their light and heavy chain subunits were investigated in digitonin-permeabilized adrenal chromaffin cells. Because these cells are permeable to proteins, the toxin had direct access to the cell interior. Botulinum type A neurotoxin and its light chain subunit inhibited Ca2+-dependent catecholamine secretion in a dose-dependent manner. The heavy chain subunit had no effect. Inhibition required introduction of the neurotoxin or light chain into the cell and was not seen when intact cells were incubated with these proteins. The inhibition of secretion by type A neurotoxin and light chain was incomplete, the maximal response being 65%. The inhibition was not overcome by increasing Ca2+ concentrations. The action of the light chain was irreversible and rapid. Botulinum type E neurotoxin also inhibited secretion in a dose-dependent manner. Its potency was increased 30-fold following mild trypsinization, which nicked the single chain protein to the dichain form. In contrast to the results seen with types A and E, botulinum type B neurotoxin did not inhibit secretion, while its light chain totally abolished secretion. Trypsinization of the neurotoxin produced the dichain form, which did not inhibit secretion. Reduction of the trypsinized neurotoxin with dithiothreitol produced inhibition equivalent to that seen with the purified light chain subunit. Isolated type A heavy chain had no effect on the inhibitory action of type A or B light chains. The data demonstrate that the ability of botulinum neurotoxins to inhibit secretion is confined to the light chain region of these proteins. Furthermore, while the botulinum neurotoxin types A, B, and E have similar macrostructures, they are not identical with respect to their biological activities.     

Characterization of the subunits of botulinum neurotoxin type A

A comparative amino acid analysis of botulinum neurotoxin type A and its subunits has been carried out. The heavy and light chains of neurotoxin have the same ratios of polar and non-polar amino acids (1.3:1), the amount of tryptophan residues in the heavy chain is 4 times as much as that in the light chain, and the number of SH-groups exceeds that in the light chains 2-fold. In neurotoxin, two N-terminal amino acid residues--alanine and leucine--were identified. Alanine was found to be the N-terminus of the heavy chain. The fluorescence spectra of neurotoxin subunits indicate differences in the conformational state of the polypeptide chains. The antigenic non-identity of botulinum neurotoxin A subunits suggests the presence in the neurotoxin molecule of at least two antigenic determinants, corresponding to the heavy and light chains.     

Dichain structure of botulinum neurotoxin: identification of cleavage sites in types C, D, and F neurotoxin molecules

Botulinum neurotoxin (NT) is synthesized by Clostridium botulinum as about a 150-kDa single-chain polypeptide. Posttranslational modification by bacterial or exogenous proteases yielded dichain structure which formed a disulfide loop connecting a 50-kDa light chain (Lc) and 100-kDa heavy chain (Hc). We determined amino acid sequences around cleavage sites in the loop region of botulinum NTs produced by type C strain Stockholm, type D strain CB16, and type F strain Oslo by analysis of the C-terminal sequence of Lc and the N-terminal sequence of Hc. Cleavage was found at one or two sites at Arg444/Ser445 and Lys449/Thr450 for type C, and Lys442/Asn443 and Arg445/Asp446 for type D, respectively. In culture fluid of mildly proteolytic strains of type C and D, therefore, NT exists as a mixture of at least three forms of nicked dichain molecules. The NT of type F proteolytic strain Oslo showed the Arg435 as a C-terminal residue of Lc and Ala440 as an N-terminal residue of Hc, indicating that the bacterial protease cuts twice (Arg435/Lys436 and Lys439/Ala440), with excision of four amino acid residues. The location of cleavage and number of amino acid residue excisions in the loop region could be explained by the degree of exposure of amino acid residues on the surface of the molecule, which was predicted as surface probability from the amino acid sequence. In addition, the observed correlation may also be adapted to the cleavage sites of the other botulinum toxin types, A, B, E, and G.     

Partial amino acid sequence of the heavy and light chains of botulinum neurotoxin type A

The dichain (nicked) type A botulinum neurotoxin is a protein (mol. wt. 145,000) composed of a heavy and a light chain (mol. wt. 97,000 and 53,000, respectively) that are held together by disulfide bond(s). We report here the sequence of the first 17 amino acid residues of the light chain, and the first 10 residues of the heavy chain. The heavy chain was isolated from the neurotoxin by two different methods, while the light chain was isolated by the only available method. The identical amino acid sequence was found in both preparations of heavy chain. Two samples of the light chain isolated from two separately prepared batches of the neurotoxin also had identical sequences.     

The complete amino acid sequence of the Clostridium botulinum type-E neurotoxin, derived by nucleotide-sequence analysis of the encoding gene.

The entire structural gene of the Clostridium botulinum NCTC 11219 type-E neurotoxin (BoNT/E) has been cloned as five overlapping DNA fragments, generated by polymerase chain reaction (PCR). Analysis of triplicate clones of each fragment, derived from three independent PCR, has allowed the derivation of the entire nucleotide sequence of the BoNT/E gene. Translation of the sequence has shown BoNT/E to consist of 1252 amino acids and, as such, represents the smallest BoNT characterised to date. The light chain of the toxin exhibits the highest level of sequence similarity to tetanus toxin (TeTx, 40%). The light chains of BoNT/A and BoNT/D share 33% similarity with BoNT/E, while BoNT/C exhibits 32% similarity. In contrast, the TeTx heavy chain exhibits the lowest degree of similarity (35%) with BoNT/E, with the BoNT heavy chains sharing 46%, 36% and 37%, for neurotoxin types A, C and D, respectively. Comparisons with partial amino acid sequences of the light chain of BoNT/E from C. botulinum strain Beluga and that from the strains Mashike, Iwanai and Otaru, indicate single amino acid differences in each case. Alignment of all characterised neurotoxin sequences (BoNT/A, BoNT/C, BoNT/D, BoNT/E and TeTx) shows them to be composed of highly conserved amino acid domains interspersed with amino acid tracts exhibiting little overall similarity. The most divergent region corresponds to the extreme COOH-terminus of each toxin, which may reflect differences in specificity of binding to neurone acceptor sites.     

Immuno-crossreactivity between botulinum neurotoxin type C1 or D and exoenzyme C3

Botulinum neurotoxin type D and exoenzyme C3 have been separately purified from Clostridium botulinum strain D-1873 to apparent homogeneity. Both ADP-ribosylated a rat liver cytosolic protein of 24 kDa. The N-terminal amino acid sequence of C3 was determined and showed a low degree of homology with those of the light and heavy chains of neurotoxins of various types which have been reported previously. However, a polyclonal antibody raised against C3 cross-reacted with the light chains, but not with the heavy chains, of type C1 and D neurotoxins. Furthermore, a monoclonal antibody recognizing the light chains of type C1 and D neurotoxins interacted with C3. These results suggest that the light chain of type C1 or D neurotoxin and exoenzyme C3 share at least one epitope in common with each other.     

Lipopolysaccharide-sensitive serine-protease zymogen (factor C) of horseshoe crab hemocytes. Identification and alignment of proteolytic fragments produced during the activation show that it is a novel type of serine protease

The horseshoe crab clotting factor, factor C, present in the hemocytes is a serine-protease zymogen activated with lipopolysaccharide. It is a two-chain glycoprotein (Mr = 123,000) composed of a heavy chain (Mr = 80,000) and a light chain (Mr = 43,000) [T. Nakamura et al. (1986) Eur. J. Biochem. 154, 511-521]. In our continued study of this zymogen, we have now also found a single-chain form of factor C (Mr = 123,000) in the hemocyte lysate. The heavy chain had the NH2-terminal sequence of Ser-Gly-Val-Asp-, consistent with that of the single-chain factor C, indicating that the heavy chain is derived from the NH2-terminal part of the molecule. The light chain had an NH2-terminal sequence of Ser-Ser-Gln-Pro-. Incubation of the two-chain zymogen with lipopolysaccharide resulted in the cleavage of a Phe-Ile bond between residues 72 and 73 of the light chain. Concomitant with this cleavage, the A (72 amino acid residues) and B chains derived from the light chain were formed. The complete amino acid sequence of the A chain was determined by automated Edman degradation. The A chain contained a typical segment which is similar in sequence to a family of repeats in human beta 2-glycoprotein I, complement factors B, protein H, C4b-binding protein, and coagulation factor XIII b subunit. The NH2-terminal sequence of the B chain was Ile-Trp-Asn-Gly-. This chain contained the serine-active site sequence-Asp-Ala-Cys-Ser-Gly-Asp-Ser-Gly-Gly-Pro-. These results indicate that horseshoe crab factor C exists in the hemocytes in a single-chain zymogen form and is converted to an active serine protease by hydrolysis of a specific Phe-Ile peptide bond.     

Calcein permeability of liposomes mediated by type A botulinum neurotoxin and its light and heavy chains

The mode of botulinum neurotoxin action involves binding of its heavy chain for internalization into the presynaptic end of a nerve cell through endocytosis. The low-pH conditions of endosomes trigger translocation of the light chain across the endosomal membrane to the cytosol, where the light chain cleaves specific target proteins involved in the docking and fusion of synaptic vesicles for acetylcholine release. In an effort to model the interaction of botulinum neurotoxin and its subunit chains with lipid bilayer at low pH during the translocation process, we have examined type A botulinum neurotoxin-mediated calcein release from asolectin liposomes. At equimolar concentration (0.1 M), the neurotoxin and its heavy and light chains evoked 23%, 58%, and 28% calcein release, respectively. Calcein release was observed only when the cis-side (the side to which neurotoxin samples were added) pH was lowered to 4. Calcein release activity of the heavy chain was mostly blocked (76%) by a polyclonal antibody raised against the neurotoxin. Additionally, two peptide-specific polyclonal antibodies derived from the N-terminal and C-terminal halves of the heavy chain were also able to block the calcein release activity by 15–20%. In summary, these results suggest that calcein release from liposomes is specifically mediated by the heavy chain, and the light chain also integrates into the membrane. Implications of these results for the molecular mode of neurotoxin light-chain translocation across the endosomal membrane are discussed.     

Amino acid sequences of the human kidney cathepsins H and L

The complete amino acid sequences of human kidney cathepsin H (EC 3.4.22.16) and human kidney cathepsin L (EC 3.4.22.15) were determined. Cathepsin H contains 230 residues and has an Mr of 25116. The sequence was obtained by sequencing the light, heavy and mini chain and the peptides produced by cyanogen bromide cleavage of the single-chain form of the enzyme. The glycosylated mini chain is a proteolytic fragment of the propeptide of cathepsin H. Human cathepsin L has 217 amino acid residues and an Mr of 23720. Its amino acid sequence was deduced from N-terminal sequences of the heavy and light chains and from the sequences of cyanogen bromide fragments of the heavy chain. The fragments were aligned by comparison with known sequences of cathepsins H and L from other species. Cathepsins H and L exhibit a high degree of sequence homology to cathepsin B (EC 3.4.22.1) and other cysteine proteinases of the papain superfamily.     

Is formation of visible channels in a phospholipid bilayer by botulinum neurotoxin type B sensitive to its disulfide?

Botulinum neurotoxin, produced by Clostridium botulinum as a approximately 150-kDa single-chain protein, is nicked proteolytically either endogenously or exogenously. The approximately 50- and approximately 100-kDa chains of the dichain molecule remain held together by an interchain disulfide bridge and noncovalent interactions. The neurotoxin binds to receptors of the target cell and is internalized by endocytosis. Thereafter, a portion of the neurotoxin, the approximately 50-kDa chain, escapes to the cytosol, where it blocks neurotransmitter release. Botulinum neurotoxin serotype B is released by the bacteria primarily as an unnicked single chain. We reduced this unnicked protein and used its binding to ganglioside in a lipid layer to produce helical tubular crystals of unnicked botulinum neurotoxin type B in its disulfide-reduced state. The helical arrangement of the neurotoxin allowed determination of the structure of the molecule using cryo-electron microscopy and image processing. The resulting model reveals that neurotoxin molecules formed loops extending out from the surface of the bilayer and bending toward a neighboring loop. Although channels have been seen with disulfide-linked neurotoxin (Schmid, Robinson, and DasGupta (1993) Direct visualization of botulinum neurotoxin-induced channels in phospholipid vesicles, Nature 364, 827-830), no channels were seen here, a finding which suggests that the reduced, unnicked neurotoxin is incapable of forming a visible channel.     

C. botulinum neurotoxin types A and E: isolated light chain breaks down into two fragments. Comparison of their amino acid sequences with tetanus neurotoxin

The flaccid paralysis in the neuromuscular disease botulism appears to depend on the coordinated roles of the approximately 50 kDa light and approximately 100 kDa heavy chain subunits of the approximately 150 kDa neurotoxic protein produced by Clostridium botulinum (J. Biol. Chem. (1987) 262, 2660 and Eur. J. Biochem. (1988) 177, 683). We observed that the light chain after separation from its conjugate heavy chain, in the presence of dithiothreitol and 2 M urea, begins to split into approximately 28 and approximately 18 kDa fragments. The other subunit-the approximately 100 kDa heavy chain following its isolation-and the parent approximately 150 kDa dichain neurotoxin do not break down under comparable conditions. This cleavage was examined in the neurotoxin serotypes A and E. The cleavage does not appear to be due to a protease. Partial amino acid sequences established that: i) the approximately 28-kDa and approximately 18-kDa fragments comprise the N- and C-terminal regions of the light chain, respectively; ii) the light chain of the neurotoxin serotypes A and E break down at precise peptide bonds; iii) the peptide bonds cleaved in serotypes A and E are five residues apart; and iv) the portions of the approximately 18 kDa fragments of serotype A and E neurotoxin sequenced so far are highly homologous to the corresponding region of tetanus neurotoxin produced by Clostridium tetani. The partial N-terminal sequence of the approximately 28 kDa fragment matches with the N-terminal sequence of the intact L chain. The 47 residues of the approximately 18-kDa fragment of type A sequenced from its N-terminal are: -Y.E.M.S.G.L.E.V.S.F.E.E.L.R.T.F.G.G.H.D.A.K.F.I.D.S.L.Q.E.N.E.F.R.L.Y.Y .Y. N.K.F.K. D.I.A.S.T.L.-. These align with those of tetanus neurotoxin beginning at its residue #259 (Tyr); the 18 underlined residues of the above 47 residues (i.e. 38%) are identical in positions between the two proteins. The 41 residues sequenced from the approximately 18 kDa fragment of type E botulinum neurotoxin are: -K.G.I.N.I.E.E.F.L. T.F.G.N.N.D.L.N.I.I.T.V.A.Q.Y.N.D.I.Y.T.N.L.L.N.D.Y.R. K.I.A.X.K. L.-.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presumptive identification of common adenovirus serotypes by the development of differential cytopathic effects in the human lung carcinoma (A549) cell culture

Abstract The neurotoxin gene from Clostridium barati ATCC43756 was cloned as a series of overlapping polymerase chain reaction (PCR) generated fragments using primers designed to conserve toxin sequences previously published. The toxin gene has an open reading frame (ORF) of 1268 amino acids giving a calculated molecular mass of 141049 Da. The sequence identity between the C. barati ATCC43756 and non-proteolytic C. botulinum 202F neurotoxins is 64.2% for the light chain and 73.6% for the heavy chain. This is much lower than reported identities for the type E neurotoxins from C. botulinum and C. butyricum (96% identity between light chains and 98.8% between the heavy chains). Previously identified conserved regions in other botulinal neurotoxins were also conserved in that of C. barati . An ORF upstream of the toxin coding region was revealed. This shows strong homology to the 3' end of the gene coding for the nontoxic-nonhemagglutinin (NTNH) component of the progenitor toxin from C. botulinum type C neurotoxin.     

Nature of intracellular type A botulinum neurotoxin.

The neurotoxin in cells of young Clostridium botulinum type A culture was extracted with lysozyme. Highly purified neurotoxin preparation, obtained by processing the extract in two chromatographic steps had only unnicked (single-chain) molecules of molecular weight comparable to that of the dichains isolated from type A crystals. Trypsinization converted the unnicked molecules into dichains whose component subunits were of sizes indistinguishable from those of the neurotoxin from crystals. The enzymatic treatment increased toxicity of crude extract 30-fold but did not activate the purified intracellular neurotoxin preparation. The results indicated that intracellular type A botulinum neurotoxin is unnicked, is not fully activated, and is activated in the time between its extraction and purification. Since trypsinization nicked all of the single chains without increasing toxicity, nicking was not causally related to toxicity activation.     

Botulinum neurotoxin type E fragmented with endoproteinase Lys-C reveals the site trypsin nicks and homology with tetanus neurotoxin

Botulinum neurotoxin type E, a 150 kDa single chain protein, cleaved with endoproteinase Lys-C yielded 113, 73, and 50 kDa fragments. The N-terminal sequence of the 113 kDa fragment, Gly-Ile-Arg-Lys-Ser-Ile-Cys-Ile, overlaps the N-terminal sequence, Lys-Ser-Ile-Cys-Ile, of the 103 kDa heavy chain produced by nicking the neurotoxin with trypsin. The -Arg-Lys- bond is therefore the site on the single chain type E NT where trypsin nicks generating the 50 kDa light and 103 kDa heavy chains of the dichain NT. The sequence of the first 50 N-terminal residues of the 73 kDa fragment were determined. This fragment is a segment of the heavy chain; 50% of the 50 residues are present in identical positions in a similar segment of the heavy chain of tetanus neurotoxin.     

Role of arginine residues in the structure and biological activity of botulinum neurotoxin types A and E

When nicked types A and E as well as the unnicked (i.e., single chain) type E botulinum neurotoxins were treated with 1,2-cyclo-hexanedione, which specifically modifies the arginine residues in 0.2 M borate buffer, pH 8.0 i) both the nicked and unnicked neurotoxins were detoxified, ii) the unnicked single chain neurotoxin became resistant to nicking with trypsin, and iii) the serological reactivity of type A (type E was not tested) was altered. Reversal of the arginine modification partially restored toxicity. In the electroimmunodiffusion test the modified type A neurotoxin appeared as 2 cones; the height of one cone increased and the other decreased as the modification reaction progressed. These results indicate that i) at least one arginine residue is involved in maintaining the toxigenic structure of types A and E neurotoxins; ii) the site of nicking in type E is an arginyl bond; and iii) arginine residue is critical for at least one antigenic determinant of type A neurotoxin.     

Covalent structure of botulinum neurotoxin type A: Location of sulfhydryl groups, and disulfide bridges and identification of C-termini of light and heavy chains

Botulinum neurotoxin Type A is synthesized byClostridium botulinum as a 150 kD single chain polypeptide. The posttranslational processing of the 1296 amino acid residue long gene product involves removal of the initiating methionine, formation of disulfide bridges, and limited proteolysis (nicking) by the bacterial protease(s). The mature dichain neurotoxin is made of a 50-kD light chain and a 100-kD heavy chain connected by a disulfide bridge. DNA derived amino acid sequencepredicted a total of 9 Cys residues (Binzet al., 1990,J. Biol. Chem. 265, 9153–9158; Thompsonet al., 1990,Eur. J. Biochem. 189, 73–81). Treatment of the dichain neurotoxin, dissolved in 6 M guanidine. HCl, with 4-vinylpyridine converted 5 Cys residues into S-pyridylethyl cysteine residues; but alkylation after mercaptolysis converted all 9 Cys residues in the S-pyridylethylated form. After confirming the predicted number of Cys residues by amino acid analysis, the positions of the 5 Cys residues carrying sulfhydryl groups and the 4 involved in disulfide bridges were determined by comparing the elution patterns in reversed-phase HPLC of the cyanogen bromide mixtures of the exclusively alkylated and the mercaptolyzed-alkylated neurotoxin. The chromatographically isolated components were identified by N-terminal amino acid sequence analysis. The HPLC patterns showed characteristic differences. The Cys residuespredicted in positions 133, 164, 790, 966, and 1059 were found in the sulfhydryl form; Cys 429 and 453 were found disulfide-bridged connecting the light and heavy chains, and Cys 1234 and 1279 were found in an intrachain disulfide-bridge near the C-terminus in the heavy chain. Ten amino acid residues, Thr 438-Lys 447,predicted to be present in the single chain neurotoxin were not found in the dichain neurotoxin. Nicking of single-chain neurotoxin by the protease(s) endogenous to the bacteria therefore appears to excise these 10 amino acid residues from the nicking region which leaves Lys 437 as the C-terminus of the light chain and Ala 448 as the N-terminus of the heavy chain. The N-terminal Pro 1 and C-terminal Leu 1295,predicted from the nucleotide sequence, remain conserved after nicking. Residues Pro 1-Lys 437 and Ala 448-Leu 1295 constitute the light and heavy chains, respectively. The C-termini were determined by isolation of short C-terminal peptide fragments and subsequent sequence analysis by Edman degradation. About 20% of the amino acid sequence predicted from DNA analysis was confirmed in these studies by protein-chemical methods.     

Human low-molecular-weight urinary urokinase. Partial characterization and preliminary sequence data of the two polypeptide chains

Low-molecular-weight urokinase (molecular weight 33100) was separated by analytical and preparative isoelectric focusing into five major subforms with isoelectric points between 8.7 and 9.6. These subforms are very similar in molecular weight, specific activity, amino acid composition and content of amino sugar and their N-terminal sequence constellation is identical. Low-molecular-weight urokinase consists of two polypeptide chains connected by a single disulfide bridge. The N-terminal region of the heavy chain (calculated Mr 30700) exhibits homology within the first 46 residues analyzed, with the known primary structure of other serine proteases. The mini chain (Mr 2426), whose complete sequence was determined, consists of 21 residues which show homology with the primary structure of the C-terminal region of the plasmin heavy chain. Based on sequence data and homology criteria with serine proteases a single-chain urokinase precursor is postulated having a peptide bond constellation between heavy and light chain region compatible with the requirements for serine protease activation.